Live attenuated viruses were the first immunization agents available for protection against viral infection. Eradication of smallpox has been achieved through widespread immunization with vaccinia virus and a similar success with poliomyelitis may be imminent through the use of the live attenuated Sabin vaccine strains.
The live attenuated vaccine strains of poliovirus were the result of serial passages in cultured cells derived from a variety of hosts (Gromeier et al, Proc. Natl. Acad. Sci. USA 93:2370-2375 (1996)). Elucidation of the genetic basis of attenuation of poliovirus neurovirulence and a better understanding of the pathogenesis of poliomyelitis have opened the possibility to derive attenuated poliovirus variants through genetic engineering (Agol et al, J. Biotechnol. 44:119-128 (1996), Almond et al, Dev. Biol. Stand. 78:161-169 (1993), Gromeier et al, Proc. Natl. Acad. Sci. USA 93:2370-2375 (1996)). Attempts to construct live attenuated polioviruses were not limited to agents for the prophylaxis of poliomyelitis. Rather, the advantageous properties of live attenuated polioviruses have inspired investigations into possible uses as immunization vectors against infectious disease other than poliomyelitis (Andino et al, Science 265:1448-1451 (1994)).
Various strategies have been employed to engineer picornavirus-based expression vectors (FIG. 1). Insertion of peptide sequences into the coding region for the viral capsid proteins was designed to display foreign immunogenic peptides on the viral capsid exterior (FIG. 1B; Arnold et al, Intervirology 39:72-78 (1996)). Dicistronic vectors were generated through insertion of foreign sequences under translational control of a secondary, heterologous IRES element inserted in between P1 and P2 (FIG. 1C) or at the N-terminus of the polyprotein (Alexander et al, Proc. Natl. Acad. Sci. USA 91:1406-1410 (1994)); FIG. 1D). Similarly, polyprotein fusion vectors were created by inserting foreign ORFs (open reading frames) into similar positions, either separating P1 from P2, or through N-terminal fusion (FIGS. 1E, 1F). Finally, poliovirus replicons were generated by replacing the coding region for the capsid proteins (P1) with a heterologous ORF (FIG. 1G).
The size of foreign gene products to be expressed varied with the strategy chosen. Minimal insertions consisting of few amino acids within the capsid (FIG. 1B) and maximum ORFs coding for gene products up to 440 amino acids in length (FIG. 1E, 1F) constitute the range of permissible insertions. It is believed that this size constraint is largely a reflection of the limited ability of the compact picornaviral capsid to accommodate genomic RNAs containing added sequences (Alexander et al, Proc. Natl. Acad. Sci. USA 91:1406-1410 (1994), Andino et al, Science 265:1448-1451 (1994)).
A major obstacle common to all proposed replicating picornavirus expression vectors is their inherent genetic instability. Picornaviruses, due to the high error rate of their RNA-dependent RNA polymerase, replicate “at the threshold of error catastrophe” (Eigen et al, RNA Genetics, eds. Domingo et al, CRC, Boca Raton, Fla., pps. 211-245 (1988)). High mutation rates create a delicate balance between beneficial rapid adaptation to changing growth environments and the limits of genetic variability imposing loss of viability. Picornaviruses evolved to maintain this balance by limiting the size of their genome (approximately 7,500 bp; Kitamura et al, Nature 291:547-553)), highly productive genome replication, and through intra- and intergenomic recombination (Wimmer et al, Ann. Rev. Genet. 27:353-436 (1993)).
Differences in the structural context and insertion locale of foreign open reading frames can have profound influences on virus propagation efficiency and, thus, expression of inserted sequences. However, irrespective of their genetic structure, all proposed expression vectors share the inherent tendency to revert to wild-type sequences with maximal propagation potential. This tendency may be due to the deleterious effect of insertion of foreign sequences on virus replication efficiency, triggering events to adapt to a faster growing phenotype. These events will invariably lead to the elimination of all or parts of the inserted foreign sequences. This has been thoroughly documented for poliovirus polyprotein fusion expression vectors (see FIG. 1F; Mueller et al, J. Virol. 72:20-31 (1998)). It was proposed that homologous recombination events lead to very rapid elimination of inserted sequences within few replicative cycles (Mueller et al, J. Virol. 72:20-31 (1998)). Frequently, the presence of minimal truncated remnants of the insert could be demonstrated for extended numbers of passages (Mueller et al, J. Virol. 72:20-31 (1998)).
Genetic instability of viral expression vectors (particularly picornavirus expression vectors) greatly limits their usefulness for vaccination purposes. Rapid deletion of inserted foreign ORFs upon virus replication diminishes expression of the immunogen. Deletion events in attenuated expression constructs can also give rise to variants displaying pathogenic properties. Genetically unstable expression vectors can be difficult to propagate on a large scale and the verification of the genotype of produced stock is a major challenge, due to the heterogeneous mixture of deletion variants generated.
The present invention results from the development of a novel strategy for engineering viral-based expression vectors, particularly picornavirus-based expression vectors. This strategy is based principally on the concept of forcing viruses to retain foreign encoding sequences by substituting the foreign sequences for regulatory sequences in a manner such that the regulatory function is retained.